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Article
N‑Acylamino Saccharin as an Emerging Cysteine-Directed Covalent
Warhead and Its Application in the Identification of Novel FBPase
Inhibitors toward Glucose Reduction
Wuqiang Wen,∥ Hongxuan Cao,∥ Yixiang Xu,∥ Yanliang Ren,* Li Rao, Xubo Shao, Han Chen, Lixia Wu,
Jiaqi Liu, Chen Su, Chao Peng, Yunyuan Huang,* and Jian Wan*
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ABSTRACT: With a resurgence of covalent drugs, there is an
urgent need for the identification of new moieties capable of
cysteine bond formation. Herein, we report on the N-acylamino
saccharin moieties capable of novel covalent reactions with
cysteine. Their utility as alternative electrophilic warheads was
demonstrated through the covalent modification of fructose-1,6bisphosphatase (FBPase), a promising target associated with cancer
and type 2 diabetes. The cocrystal structure of title compound W8
bound with FBPase unexpectedly revealed that the N-acylamino
saccharin moiety worked as an electrophile warhead that covalently
modified the noncatalytic C128 site in FBPase while releasing
saccharin, suggesting a previously undiscovered covalent reaction
mechanism of saccharin derivatives with cysteine. Treatment of title compound W8 displayed potent inhibition of glucose
production in vitro and in vivo. This newly discovered reactive warhead supplements the current repertoire of cysteine covalent
modifiers while avoiding some of the limitations generally associated with established moieties.
■
INTRODUCTION
With the development of modern medicine, targeted covalent
inhibitors (TCIs) have become greatly successful therapies for
a broad array of human diseases such as non-small-cell lung
carcinoma, mantle cell lymphoma, and type II diabetes.1−3
Currently, approximately one-third of targeted enzyme drugs
approved by the Food and Drug Administration (FDA) are
covalent inhibitors.4,5 Due to the increased strength and often
irreversible nature of the covalent bonds formed between a
TCI and target, the use of covalent inhibitors offers the
potential for increased potency and prolonged pharmacodynamics effects, compared to traditional noncovalent inhibitors.6,7 In addition, targeting disease proteins and pathways
with covalent inhibitors has become a feasible option for
overcoming drug resistance and protein mutation and
improving the protein isoform selectivity and toxicity
associated with noncovalent inhibitors.4,8−10
The TCIs bind to target proteins in two distinct necessary
steps: the first step involves the reversible binding of a highaffinity ligand to its biological target and then an electrophilic
“warhead” on the ligand binds at the appropriate position to
form a covalent bond with a nucleophilic residue on the
protein.9,11 A number of electrophilic warheads have been
explored to react with nucleophile residues, including cysteine,
lysine, or tyrosine;12,13 however, cysteine’s thiol is endowed
with enhanced reactivity, and the paucity of cysteine in the
© 2022 American Chemical Society
proteome coupled with the fact that closely related proteins do
not necessarily share a given cysteine residue enables a level of
unprecedented rational target selectivity, making cysteine the
most favored target.14,15 Warhead selection typically starts with
an estimation of the reactivity required to target the desired
amino acid, as the reactivity profiles of the covalent reactive
group (warhead) affect the target specificity of TCIs.16
Nevertheless, it is still challenging to strike the right balance
between reactivity and selectivity. The most recently reported
warheads undergo both Michael-type and non-Michael-type
nucleophilic addition, addition−elimination reaction, nucleophilic substitution, and oxidation.12,17 Targeting noncatalytic
cysteine residues with acrylamides and other α,β-unsaturated
carbonyl compounds is the “classical” strategy of TCI
development.15,18 A recent analysis of cysteine-targeted
covalent inhibitors revealed that nearly 70% of the published
compounds carried Michael acceptor-type warheads, and
acrylamide is the preponderant functional group12 due to its
Received: March 2, 2022
Published: July 5, 2022
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Figure 1. (A) Model reaction of W1 with thiol reagent 1,4-benzenedithiol. Reaction conditions: tetrahydrofuran (THF), N2, rt. (B) Model reaction
of W1 with glutathione (GSH, 1.5 equiv) in pH 7.4 D2O/d6-acetone (1/5) at 37 °C. (C) Proposed reaction mechanism of W1 with cysteine.
In this study, we report our efforts that sweetener saccharin
derivatives can be used to covalently modify the thiols or
cysteine(s) of proteins through a previously unreported
reaction mechanism. Most particular interest is that this
newly discovered warhead not only covalently inhibits target
protein but also releases saccharin, which is not metabolized
and considered safe by the FDA. Saccharin derivatives have
shown inhibitory activity against various different subtypes of
carbonic anhydrases in humans by coordinating with their zinc
ions;40,41 however, there are many zinc-dependent enzymes in
organisms,42 which makes it difficult for these inhibitors to act
effectively on their intended target. The covalent reaction of
saccharin derivatives that we newly report herein provides a
new strategy for achieving the purpose of the precise release of
saccharin inhibitors through reaction with glutathione (GSH)
or cysteine in the future. The reactivity of this novel warhead
was assessed on the basis of GSH reactivity through a
quantitative 1H NMR (qNMR) method43 and quantum
mechanical (QM) calculations. Promising scaffolds were
investigated further in the identification of the inhibitory
ability of fructose-1,6-bisphosphatase (FBPase), a key ratecontrolling enzyme in gluconeogenesis44,45 and a promising
target associated with cancer and type 2 diabetes.46,47 Our
previous studies have identified a new covalent allosteric site
(C128) of FBPase,48,49 which provides a promising way for the
design of covalent allosteric drugs for glucose reduction,
allowing incorporation of an electrophilic group at the
appropriate position. Therefore, we sought to design a novel
covalent inhibitor of FBPase with an optimal warhead. The
covalent binding mode was identified by combining sitedirected mutagenesis, protein liquid chromatography with
mass spectrometry (LC−MS), and cocrystal structure analysis.
Notably, compounds W8 and W8k exhibited high selectivity
against FBPase and W8 effectively reduced blood glucose in an
Institute of Cancer Research (ICR) mice model and dosedependent inhibition of glucose production in a primary
mouse hepatocyte model.
ease of synthesis; acceptable reactivity window with cysteine
over other amino acids; and absorption, distribution,
metabolism, excretion, toxicity (ADMET) compatibility.19
However, the majority of drugs featuring a covalent binding
modality were discovered serendipitously and were only
retrospectively identified as covalent inhibitors therefore,
only a few drugs that contain the abovementioned “classical”
electrophiles have entered clinical practice.5 Recent cytochemical proteomic studies have shown that certain types of
acrylamide-based kinase inhibitors induce the expression of offtarget protein markers in the submicromolar concentration
range.20 Therefore, there remains a significant need for
additional cysteine reactive warheads with tunable properties
and reactivity profiles.6 Similarly, many issues with other types
of established warheads remain to be resolved. For example,
with respect to nucleophilic substitution-based warheads, no
studies on sulfonyl fluorides,21,22 nucleophilic aromatic
substitution (SNAr) electrophiles,23−25 or activated esters26,27
investigated the toxic potential of the leaving group.17 In
addition, problems may arise when the target residue is poorly
reactive, difficult to access, or incompatible with the spatial and
geometric requirements of these electrophilic warhead groups.
This prompted us to search for as-yet unexplored electrophiles
to increase the electrophilic warhead options available for TCI
design.
Saccharin, is an orally effective, noncalorie artificial sweetener (NAS).28 Since saccharin is not metabolized and is
considered safe by the FDA,29 it has been used as a
substructure in a number of bioactive compounds, including
carbonic anhydrase (CA) inhibitors,30−33 leukocyte elastase
inhibitors,34 and neutrophil elastase inhibitors.29 Furthermore,
compounds containing saccharin fragments are used in the
clinic for antidepressant35 or stroke36,37 therapy. Notably,
saccharin has recently been reported to be a warhead that can
covalently bind to serine through a nucleophilic addition
reaction;38,39 however, this type of covalent reaction results in
the destruction of the saccharin structure and diminished
safety.
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Scheme 1. Synthesis of Compounds W2 and W3a
RESULTS AND DISCUSSION
Electrophilic Reactivity of W1. Saccharin-based inhibitors have been described as irreversibly acylated compounds
of rhomboids (intramembrane serine proteases), and saccharin
has been regarded as a warhead that can covalently bind to
serine through a nucleophilic addition reaction.37 Specifically, a
reaction mechanism in which the serine in the active site of a
protease attacks the carbonyl group of saccharin, leading to
opening of the ring in the saccharin-based inhibitor and the
formation of an acyl-enzyme, has been proposed. Nevertheless,
the application of this type of saccharin-based inhibitor is very
limited in drug discovery due to its low reactivity of the
endocyclic carbonyl group.
In this study, we found that the reaction of Nbenzoylsaccharin (W1) with 1,4-benzenedithiol affords saccharin and S-(4-mercaptophenyl) benzothioate (adduct 1,
Figures 1A and S1), indicating that W1 may be a potential
thiol electrophilic reagent with a novel reaction mechanism
that has never been reported. Interestingly, this reaction
releases saccharin, a sweetener that is not metabolized and
considered safe by the FDA, as well as an inhibitor of CA
linked to many diseases such as edema, glaucoma, epilepsy,
and cancer.31 Therefore, W1 may be a potential novel covalent
warhead covalently binding to the thiol of cysteine.
GSH is a tripeptide consisting of glutamic acid, cysteine, and
glycine, and it contains a free thiol moiety that acts as a
reactive nucleophilic site and has been typically applied to the
identification of electrophilic reagents in vitro.23,49,50 On the
basis of the reaction (Figure 1A) of W1 and 1,4benzenedithiol, we deduced that it is feasible to use W1 as a
novel electrophile to react with GSH, and their probable
products (Figure 1B) are Adduct 2 and saccharin. To
determine the feasibility of this reaction, the solution of W1
in acetone-d6 was treated with a solution containing 1.5 mol
equiv of GSH in pH 7.4 buffered D2O. As expected, the
saccharin was isolated from the products. Furthermore, nuclear
magnetic resonance (NMR) spectroscopy (Figure S2B) was
performed to trace Adduct 2 of this reaction. It is shown in
Figure S2B that the peaks corresponding to the methylene (a)
of GSH and the phenyl (b) of W1 decrease gradually, while
two newly formed peaks (c and d), which are related to the
products, increase gradually, suggesting the presence of
Adduct 2 proposed in Figure 1B.
On the basis of the aforementioned experimental data, we
proposed the possible reaction mechanism (Figure 1C) of W1
reacting with a thiol. This reaction starts with a nucleophilic
attack on the exocyclic carbonyl group (blue), leading to a
tetrahedral alkoxide transition state (Figure S3), which has
been predicted at the ωb97xd/6-31+g(d)-SMD level of theory
using the Gaussian 09 software package.51 The transition
energy barrier (ΔE1) of step 1 is 65.87 kcal/mol. The alkoxide
negative charge acquired greater stability after being transferred to the leaving group (saccharin), and the elimination of
the leaving group (saccharin) in step 2 allowed the carbonyl
reformation of a new acyl compound with cysteine. Our
calculated energy barrier (ΔE1) for step 2 was 75.8 kcal/mol.
Chemistry. Compound 2 was synthesized by a known
methodology.52 Then, nucleophilic substitution of 2 with
benzoyl chloride in dichloromethane (DCM) (in the presence
of Et3N) at room temperature (rt) yielded W2 (Scheme 1).
Compound W3 was prepared using the same reaction route as
that for W2. W4−W6 were synthesized using saccharin (1) as
a
Reagents and conditions: (a) THF, LiAlH4, rt, 1 h; (b) benzoyl
chloride, DCM, rt, Et3N, 1.5 h.
the starting material with the same reaction route as that for
compound W3 (Scheme 2). W7 was prepared as previously
described53 (Scheme 2). The formation of compound 8 in the
reaction of compound 7 with triphosgene was followed by a
reaction between this intermediate compound and saccharin to
afford compounds W8, W9, and W8a−W8p (Scheme 3).
Compound 9 was hydrolyzed to give compound 10 in a DCM
solution of trifluoroacetic acid (Scheme 3). Then, AP1 was
synthesized using compound 10 as the starting material with
the same reaction route as that for compound W8.
Tuning the Reactivity of the Reactive Group.
Normally, compounds with high reactivity can be easily
cleared and can generate nonspecific adducts; therefore, it is
extremely important to tune the reactivity of the electrophilic
warhead to the intended enzyme target to prevent off-target
reactivity.54,55 The intrinsic reactivity of this warhead should
be sufficient to covalently modify a specific target when the
compound is reversibly bound but not susceptible to chemical
stability, metabolic problems, or undifferentiated reactions with
other proteins.56 The half-life (t1/2) values of the compounds
reacting with buffer, dimethyl sulfoxide (DMSO, a weak
nucleophilic reagent57), and glutathione (GSH) provide useful
information about their stability, electrophilicity, and likelihood of forming reactive intermediates. In this study, the
buffer stability of the compounds was characterized by 1H
NMR-based kinetic methods in the absence of GSH. The GSH
half-life (GSH t1/2) and DMSO half-life (DMSO t1/2) were
determined through the first-order-reaction rate constant
(kpseudo‑1st). As illustrated in Figure 2B, W1 showed the
appropriate stability (>10 000 min) required for the buffer;
however, it was not sufficiently stable (DMSO t1/2 = 52.5 min)
in the presence of DMSO, which may be related to the weak
nucleophilicity of DMSO. The NMR experiment indicated that
the sulfoxide group of DMSO presumably attacks the carbonyl
group (blue) of W1, and then undergoes a rearrangement to
give the adduct and saccharin (Figure S4). These experimental
results showed that W1 was unstable in a highly electrophilic
environment, rendering it incompatible for incorporation into
druglike systems. Therefore, the systematic study of the
reactivity of various electrophiles with DMSO, GSH, and the
corresponding structure−reactivity relationship (SRR) is useful
for the development of novel covalent warheads; thereby, a
series of derivatives of W1 were synthesized to tune its
reactivity and stability.
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Scheme 2. Synthesis of Compounds W4−W7a
Reagents and conditions: (a) EtOAc, rt, Et3N, 2 h; (b) THF, 0 °C, 20 min; 50 °C, 6 h; rt, 12 h.
a
Scheme 3. Synthesis of Compounds W8, W9, and W8a−W8qa
Reagents and conditions: (a) triphosgene, DCM, rt, Et3N, 2 h; (b) saccharin, EtOAc, reflux, 1 h. (c) CF3COOH, DCM, 0 °C, 12 h.
a
negative charge particularly when it exhibits relatively high
electronegativity or delocalization with respect to the negative
charge.
Considering the contributions of carbonyls and sulfones to
the electron-withdrawing property of the leaving group
(saccharin), we attempted to synthesize compounds W2 and
W3 by replacing the carbonyl and sulfone of saccharin with
CH2 groups to reduce the rate of step 2 (Figure 2A).
Surprisingly, compounds W2 and W3 showed no reactivity
toward DMSO (t1/2 > 10 000 min) or GSH (t1/2 > 10 000
According to the proposed mechanism shown in Figure 1C,
the overall reaction rate of this type of compound with thiol
was controlled by the following two factors: the stability of the
carbonyl (step 1) and the effectiveness of the leaving group
(step 2). To our knowledge, the less partial positive charge on
the carbonyl group is conducive to more stabilization of
carbonyl; hence, the electron-donating or -withdrawing
capacity of substituents attached to the carbonyl carbon is a
primary factor affecting carbonyl stabilization. The effectiveness of a leaving group is related to its ability to stabilize a
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Figure 2. Tuning the reactivity of the electrophiles. (A) Optimization of strategies for modifying the leaving group ability. (C) Optimization of
strategies for modifying the stability of the carbonyl moiety. Evaluation of the half-time (t1/2) of the reaction between electrophiles (B) W1−W3 or
(D) W4−W9 and stability in buffer (acetone-d6/D2O = 5:1), DMSO, and GSH. The general formulas of compounds W4−W9 are shown in (C).
(E) Crystal structure of W8. CCDC 2105 353 contains the supporting crystallographic data for this paper. These data can be obtained free of
charge from The Cambridge Crystallographic Data Centre via https://www.ccdc.cam.ac.uk/. aReaction conditions: electrophile (20 μmol) in buffer
at 37 °C. bReaction conditions: electrophile (20 μmol) in DMSO-d6/D2O (5:1) at 37 °C. cMeasurement could not be obtained due to high
reactivity. dReaction conditions: electrophile (20 μmol) and GSH (30 μmol) in DMSO-d6/D2O (5:1) at 37 °C.
acceptable reactivity profiles (Figures 2D and S8). To further
investigate the effect of the nitrogen atoms on reactivity, an Xray structure of W8 was resolved. As shown in Figure 2E, an
intramolecular hydrogen bond (with a distance of N(2)−
H(2)···O(1) = 0.88 Å) between the hydrogen atom of the
amino group and the oxygen atom of the carbonyl group was
formed, which remarkably enhanced the stabilization and
reduced the reactivity of the carbonyl carbon in W8 upon
nucleophilic attack. Therefore, N-acylamino saccharin can be
regarded as a potential emerging covalent warhead for use in
designing novel covalent drugs.
Next, we synthesized the analogues of W8 by replacing the
phenyl group with different groups (Table 1), such as propyl
(W8a), thiophen-2-yl (W8b), cyclohexyl (W8c), naphthalen1-yl (W8d), and 1-phenylethyl (W8e), to explore the effect of
phenyl on the reactivity of W8. Compared with W8, most of
these derived compounds were more reactive toward GSH, the
thiophen-2-yl- and naphthalen-1-yl-containing compounds
exhibit the most reactivity (GSH t1/2 = 21.9, 23.7 min).
Furthermore, a library of W8 derivatives, including those
formed through the substitution of withdrawing groups (i.e.,
trifluoromethyl and halogen) and donating groups (i.e., methyl
and methoxy), was synthesized to evaluate the effects of the
substituents of the benzene ring on the reactivity of these
compounds. Similarly, the majority of these compounds
showed increased activity (GSH t1/2 = 11.2−47.5 min), except
W8k (GSH t1/2 = 67.3 min). Notably, the electron-donating
substituents on benzene appeared to reduce the reactivity of
the corresponding compounds; for instance, the half-lives of
compounds W8j (GSH t1/2 = 47.5 min) and W8k were longer
than those of W8f−W8i (GSH t1/2 = 11.2−35.9 min). Taken
together, these experiments suggested that the N-acylamino
saccharin set represents a modular chemotype amenable to
reactivity fine-tuning.
min) (Figure 2B). We hypothesized that the disappearance of
the W2 and W3 reactivity could be attributed to the lack of the
core moduleN-acetylated saccharin; therefore, we retained
N-acetylated saccharin in further warhead optimization
experiments. Subsequently, electron-donating alkyl substituents (W4 and W5, Figure 2C) were attached to the carbonyl
carbon to reduce the partial positive charge on the carbonyl
carbon of the side chain, making them less reactive to
nucleophilic attack than the parent compound W1. It is
noteworthy that W4 (DMSO t1/2 = 203.8 min) and W5
(DMSO t1/2 = 346.5 min) showed lower reactivity toward
DMSO than W1 (Figure 2D), suggesting that the electrondonating ability of the alkyl substituents was sufficient to
reduce the rate of step 1. Therefore, we incorporated the
greater electron-donating alkoxy groups at the X-position (W6
and W7). Compound W6 (DMSO t1/2 = 182.4 min) exhibited
a half-life in DMSO similar to that of W4, but W7 failed to
react with DMSO, suggesting that the introduction of alkoxy
groups was effective in reducing the rate of step 1. However,
W7 maintained significantly high reactivity (GSH t1/2 < 1 min)
toward GSH and was therefore further optimized through the
introduction of a greater electron-donating group.
The nitrogen atom in amides is a powerful electron-donating
group through resonance. The lone pair of electrons on a
nitrogen atom of an amide can form π bonds with a carbonyl
group, thereby reducing the reactivity of the carbonyl and
inhibiting the free rotation of the C−N bond in the amide.
Moreover, nitrogen is less electronegative than oxygen, and the
stabilization of delocalized positively charged resonance
structures is usually better than that of other acid derivatives.
Therefore, to further reduce the reactivity of the N-acetylated
saccharin warhead, W8 and W9 were synthesized by the
introduction of a nitrogen atom at the X-position. Surprisingly,
W8 (GSH t1/2 = 61.3 min) and W9 (GSH t1/2 = 33.6 min)
exhibited high stability (DMSO t1/2 > 10 000 min) and
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Table 1. Half-Life (t1/2, min) of Synthesized Compounds, Disulfiram and Afatinib, in the Presence of pH 7.4 Buffer, DMSO,
and Glutathione (GSH)
Reaction conditions: electrophile (20 μmol) in buffer at 37 °C. bReaction conditions: electrophile (20 μmol) in DMSO-d6/D2O (5:1) at 37 °C.
Reaction conditions: electrophile (20 μmol) and GSH (30 μmol) in DMSO-d6/D2O (5:1) at 37 °C.
a
c
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compounds W8a and W8 (GSH t1/2 = 61.3, 46.5 min, Table 1)
showed similar reactivity; however, FBPase inhibition of W8a
(>500 μM) was remarkably different from that of W8 (3.4
μM), indicating that the N-substituted benzyl group was
essential for the covalent inhibition of FBPase. Similar to W8,
W8e−W8m possessed an N-substituted benzyl group, thereby
showing high inhibitory activity against FBPase. In particular,
W8k exhibited the most potent inhibition with a half-maximal
inhibitory concentration (IC50) of 1.7 μM, which was similar
to that of disulfiram (1.5 μM). This evidence indicated that
the π−π stacking interaction between FBPase and N-acylamino
saccharin inhibitors may be crucial for FBPase inhibition.
As illustrated in Figure S7, the NMR experiments revealed
that the reaction mechanism of W8 with GSH was similar to
that of W1, with the formation of N-benzylthiocarbamate from
W8 and GSH accompanied by the departure of saccharin; we
thus proposed an SN-based mechanism (Figure 3A) for the
modification of cysteines in proteins by W8. The liquid
chromatography−mass spectrometry (LC−MS) experiments
of representative compound W8 and FBPase were carried out
in Figure 3B to confirm this reaction mechanism. W8 was first
preincubated with FBPase for 10 min and analyzed by mass
spectrometry for identification of potential protein−ligand
adducts (MS). As illustrated in Figure 3B, peptides (133.05
Da) of C128 covalently bound with part (N-benzyl
carboxamide fragment, W8-2) of W8 can be observed,
demonstrating that W8 could covalently modify C128 of
FBPase. In addition, only the C128A mutation led to a
significant increase (over 147-fold, Table S2) in the IC50 of
W8, compared to that of wild-type (WT) FBPase. This finding
provides new evidence for the confirmation of the newly
discovered reaction mechanism (Figure 1C) of N-acylamino
saccharin derivatives with cysteine in enzymes. Site-directed
mutation assays and LC−MS data showed that, as in our
previous studies,47 C128 is extremely important for the
covalent reaction of N-acylamino saccharin derivatives with
FBPase.
Furthermore, the cocrystal structure of the FBPase bound
with W8 (PDB ID: 7WJV, Figures 3C and S10) was resolved
to elaborate the reaction mechanism of N-acylamino saccharin
derivatives and C128 in FBPase. The electron density map of
the allosteric site indicated that the N-benzyl carboxamide
fragment (W8-2) was close to the C128 residue, and C−S
bond formation was unambiguously confirmed by the electron
density in chain b (Figure 3C). These results confirm that,
similar to W1, W8 covalently modifies the cysteine in FBPase
through the newly discovered reaction mechanism, as shown in
Figure 1C. The alignment of FBPase and W8 cocrystal
structures revealed that W8-2 occupied the allosteric site of
FBPase with a binding mode similar to that of 214b (PDB ID:
6LS5).47 Notably, the benzene ring of W8-2 also formed two
weakly π−π stacking interactions with the phenyl of residue
Y258 and imidazolyl ring of H253 (with the distances of 4.7
and 4.2 Å, respectively)61 and a cation−π stacking interaction
with the guanidine group of residue R254 (with a center
distance of 5.5 Å),62 demonstrating the importance of the
aromatic ring in the activity of the hit compounds. To identify
the central and important role of these three residues in
covalent inhibitory regulation when FBPase was covalently
modified by W8-2, the IC50 values of W8 against Y258A,
H253A, and R254A mutants were determined systematically
(Table S3). As listed in Table S3, R254A mutation led to a
remarkable increase (21.6-fold) in the IC50 of W8, compared
To evaluate the chemical stability of N-acylamino saccharin
compared with that of commercially available covalent drugs
that are known to be covalently bound to cysteine residues, the
half-lives of disulfiram and afatinib toward GSH were
determined following the aforementioned protocol. Disulfiram,
a well-known antialcoholism58 and a first-generation covalent
inhibitor of FBPase with a disulfide bond,59 yielded a GSH t1/2
value of 32.8 min. Afatinib, an irreversible tyrosine kinase
inhibitor with acrylamide,60 showed lower GSH reactivity
(GSH t1/2 = 88.8 min). Compared with disulfiram, compounds
W8b, W8d, W8g−W8i, and W8n−W8p exhibited much faster
reaction rates (GSH t1/2 = 11.2−29.5 min), while W8e, W8f,
W8l, W8m, and W8q (GSH t1/2 = 35.9−31.4 min) showed
similar reactivities. In addition, W8, W8a, W8c, W8j, and W8k
showed much slower reaction rates (GSH t1/2 = 46.5−67.3
min) than disulfiram. To gain a deeper mechanistic understanding of the reactivity of N-acylamino saccharin with
biological thiols and to explain the differences in reactivity
between similarly substituted analogues, we calculated the
lowest unoccupied molecular orbital (LUMO) energies by the
density functional theory (DFT) method, and the results are
listed in Table 1. The increase in reactivity observed when
going from W8a, W8d, and W8k to W8h, W8i, and W8n can
be explained by differences in the electronic factors.
Incorporation of an electron-withdrawing group into the
aromatic ring (W8h, W8i, W8n) resulted in the lowering of
calculated LUMO energies, thereby exhibiting high reactivity.
Notably, the strong correlation (R2 = 0.5936, Figure S5)
between GSH t1/2 and LUMO energies suggests that the Nacetylated saccharin-based warheads have tunable reactivity by
the introduction of electron-withdrawing groups into the
aromatic ring. In brief, W8 analogues with moderate to weak
reactivity (GSH t1/2 = 31.4−67.3 min) are suitable for the
development of target-specific covalent inhibitors. The tunable
GSH reactivity of N-acylamino saccharin as a novel covalent
warhead establishes a progressively stronger foundation for its
further application.
Utilization of N-Acylamino Saccharin Warheads in
FBPase Inhibitor Scaffolds. Targeting FBPase is an
emerging approach for diabetes therapy, and the C128 site
on FBPase has recently been proven to be a highly promising
strategy for designing drugs with hypoglycemic effects in vivo
and in vitro.47 To explore the application of these N-acylamino
saccharin derivatives in a druglike setting, eight compounds
(Table 2) with low reactivity toward GSH (t1/2 > 30 min) were
chosen to evaluate their FBPase inhibitory ability, binding
mode, selectivity, and in vivo effect. As listed in Table 2,
Table 2. FBPase Inhibitory Activities (IC50 μM) of W8
Analogues
compounds
IC50 (μM)a
W8
W8a
W8c
W8e
W8f
W8j
W8k
W8m
disulfiram
3.4 ± 0.5
>500
16.4 ± 1.0
8.7 ± 1.5
4.2 ± 0.1
3.6 ± 0.5
1.7 ± 0.2
1.9 ± 0.5
1.5 ± 0.3
Article
a
IC50 values were calculated from the FBPase activity assay.
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Figure 3. (A) Proposed mechanism for covalent modification of FBPase by W8. (B) Mass spectra of FBPase preincubated with W8-2 (predicted
Δm, 133.05 Da; found Δm, 133.05 Da). (C) X-ray cocrystal structure of W8-2 and FBPase (PDB ID: 7WJV) (2Fo−Fc omit map contoured at
0.8σ).
listed in Table S5, all six compounds exhibited high selectivity
(>25-fold) for ALDOs (ALODA, ALDOB, and ALDOC), and
three compounds (W8, W8k, and W8m) displayed high
GAPDH selectivity (>25-fold). In comparison, W8f showed
moderate GAPDH selectivity (16.3-fold). No remarkable
GAPDH selectivity was observed for W8e (1.3-fold) and
W8j (3.6-fold), as shown in Table S5. Taken together,
compounds W8 and W8k exhibited not only high ALDO
selectivity (>40-fold) but also high GAPDH selectivity (>27fold). Furthermore, we used the known multitarget covalent
inhibitor disulfiram as the control and determined its
inhibitory activity against four enzymes. As shown in Tables
1 and 2, the reactivity and inhibitory activity against FBPase of
disulfiram (GSH t1/2 = 32.8 min, IC50 = 1.5 μM) were similar
to those of W8f (GSH t1/2 = 35.9 min, IC50 = 4.2 μM).
However, inhibitory activities of disulfiram against four
enzymes (ALDOA, ALDOB, ALDOC, GAPDH) were almost
higher than those of W8f (Table S5), indicating that the Nacylamino saccharin warhead provided in this work has some
selectivity over disulfiram.
to that of WT FBPase, but Y258A and H253A mutations show
similar inhibition to WT FBPase. These results indicated that
the π−π stacking interactions between W8 and Y258/H253
were weakly possible due to the longer distances between
them. In comparison, the cation−π stacking interaction
between W8 and H253 was nontrivial for the covalent binding
of W8 against FBPase. In conclusion, these findings not only
confirmed the covalent binding of W8 to C128 but also more
clearly showed the mechanism of the reaction between Nacylamino saccharin derivatives and cysteine residues.
Target selectivity is a key but challenging issue in the design
of safe and effective covalent ligands due to concerns about the
formation of nonspecific or untargeted adducts that lead to
potential toxicity. To evaluate the selectivity of the Nacylamino saccharin warhead on the targets in the glucose
metabolic pathway, the inhibitory activities of hit compounds
(i.e., W8, W8e, W8f, W8j, W8k, and W8m) against several
essential targets in the glucose metabolic pathway (i.e.,
ALDOA, ALDOB, ALDOC, and glyceraldehyde 3-phosphate
dehydrogenase (GAPDH)) were evaluated (Table S5). As
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Figure 4. Effect of a single administration of compound W8 on blood glucose in 7−9 week-old ICR mice. (A) Blood glucose changes and (B) AUC
of blood glucose between 0 and 6 h in 12 h fasted ICR mice after oral administration of compound W8 (n = 5−6 in each group). (C) Blood
glucose responses to an oral glucose challenge (2 g/kg) and (D) AUC of blood glucose between 0 and 2 h in 12 h fasted ICR mice. Compound W8
was intraperitoneally administered 1 h prior to the oral glucose challenge. (E) Effects of W8 on glucose output in primary rat hepatocytes after
overnight serum starvation treatment (LG-DMEM) at concentrations of 50−300 μM. (F) Relative protein expression of primary rat hepatocytes in
physical conditions after treatment by compounds W8 at concentrations above 50−300 μM. Data are presented as the mean ± standard error of
the mean (SEM) (*P < 0.05, **P < 0.01, ***P < 0.001 vs vehicle; Student’s t-test). Metformin, Met.
to 130 kDa. In particular, 2 μM AP1 could predominantly label
a 35 kDa protein of the hepatic LO2 cell, as illustrated in
Figure S11C, indicating the better selectivity of AP1 in
covalent binding with proteins of the LO2 cell. These
experimental results further suggested that the N-acylamino
saccharin warhead exhibited good selectivity in the LO2 cell.
Glucose Reduction in ICR Mice. Our aforementioned
experiments demonstrated that N-acylamino saccharin derivatives covalently bound to the C128 site of FBPase to inhibit
FBPase enzymatic activity, which encouraged us to further
evaluate their glucose-lowering effects in vivo and to
investigate the applicability of these covalent warheads in
promoting hypoglycemia. Considering their superior FBPase
inhibition, compounds W8 and W8k were chosen to evaluate
their potency in blood glucose management and the inhibition
of glucose output in mouse primary hepatocytes. First, ICR
mice were intraperitoneally administered 30 mg/kg saccharin,
W8, or W8k; and then, blood glucose was measured during the
following 0−6 h period. As shown in Figure S12A,B, 30 mg/kg
W8 exhibited an apparent glucose-lowering effect at 2 and 6 h.
In comparison, saccharin administered at a concentration of 30
mg/kg, the dose equivalent to that of W8, upon covalently
reacting with FBPase, reduced the glucose level, showing no
significantly glucose-lowering effect during the 0−6 h period.
These results preliminarily indicated that W8 but not
saccharin exerted a glucose-lowering effect on the mice
model because W8 could covalently bind to FBPase. Notably,
30 mg/kg W8k failed to display a hypoglycemic effect, which
may be due to its poor water solubility, which makes it difficult
to effectively target hepatic FBPase in vivo.
In addition, the inhibitory activities of compounds W8 and
W8k against three typical kinases (Bruton’s tyrosine kinase
(BTK), epidermal growth factor receptor (EGFR), and Janus
kinase 3 (JAK3)), which are common drug targets for cysteine
covalent inhibition, were analyzed to assess the selectivity of
this type of novel covalent warhead. As shown in Table S6,
compounds W8 and W8k exhibited very low inhibition
activities in BTK/EGFR/JAK3 (300 μM inhibition rate is
less than 50%), indicating that they existed more than 100-fold
selectivity between FBPase and BTK/EGFR/JAK3. Therefore,
these two N-acylamino saccharin compounds showed a high
probability of usefulness in the design of covalent inhibitors
against FBPase.
Activity-based protein profiling (ABPP) is one of the
chemical proteomic approaches that use small-molecule
chemical probes to understand the interaction mechanisms
between compounds and targets, which can be used to identify
the protein targets of small molecules and even the active sites
of target proteins. Thus, to assess the proteomic reactivity of
compound W8, we designed and synthesized the ABPP probe
AP1 based on the structure of W8. Iodoacetamide alkyne
(IAA), a well-known nonselective covalent probe, was used as
the control. ABPP was performed in hepatic LO2 cells or
purified FBPase in terms of the workflow shown in Figure
S11A. As illustrated in Figure S11B, AP1 could label the
purified FBPase of about 35 kDa in a dose-dependent manner.
It should be noticed from Figure S11C that 2−8 μM AP1
could selectively label 35 kDa (the molecular weight of
FBPase) and 40 kDa proteins of the hepatic LO2 cell; in
comparison, 2 μM IAA could label almost all proteins from 15
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warheads in drug molecules. The weak reactivity and better
target selectivity of N-acylamino saccharin warheads were
successfully exploited for the development of covalent
inhibitors, and the utility of these novel electrophilic warheads
as chemical biological probes was demonstrated through the
covalent modification of FBPase, a promising target associated
with cancer and type 2 diabetes. The cocrystal structure of
compound W8 reacting with FBPase unexpectedly revealed
that the N-acylamino saccharin moiety was an electrophile
warhead that covalently modified the noncatalytic C128 site of
FBPase, and the released saccharin was observed to be nearby
the C128 site, suggesting a previously unrecognized covalent
reaction mechanism of saccharin derivatives. Moreover, a
cation−π stacking interaction between the benzene ring of W8
and the guanidine group of surrounding residue R254 was
nontrivial for the covalently binding of W8 against FBPase.
Notably, treatment with compounds W8 reduced blood
glucose in an ICR mice model, as well as led to dosedependent (50−300 μM) inhibition of glucose production
under physiological conditions (P < 0.001 vs vehicle) with low
toxicity. Overall, this type of novel warhead with desirable
reactivity and stability profiles is easily synthesized and,
therefore, is an appropriate supplement to the current
repertoire of cysteine covalent modifiers that lack some of
the limitations generally associated with established moieties.
Notably, a product of this novel covalent reaction is saccharin
or a saccharin derivative, which has been proven to be a
carbonic anhydrase (CA) inhibitor, hence alternatively
suggesting a new strategy for leveraging the protective effect
and targeted release of saccharin drugs in the future.
Second, ICR mice were also intraperitoneally administered
30 and 10 mg/kg W8 for a detailed evaluation of its
hypoglycemic effect. As illustrated in Figure 4A,B, 10 and 30
mg/kg W8 showed significant glucose-lowering effects at 2 h,
and the area under the curve (AUC)0−6h analysis revealed that
10 and 30 mg/kg W8 resulted in 15.3% and 42.4% reduction in
blood glucose, respectively.
Third, an oral glucose tolerance test (OGTT) was
performed to explore the glucose tolerance capacity of the
lead compound. Figure 4C,D shows that the blood glucose in
the control group dramatically increased to 13.34 mM 0.25 h
after oral 2 g/kg glucose intake, while the 10 and 30 mg/kg
treated groups exhibited a significant suppressive effect on
blood glucose. The AUC0−2h analysis also suggested that the
10 and 30 mg/kg treated groups showed reductions in glucose
of 20.2% and 17.7%, respectively. In summary, these in vivo
experiments clearly demonstrated that W8, an N-acylamino
saccharin derivative, exerted a glucose-lowering effect on ICR
mice.
Inhibition of Glucose Output in Mouse Primary
Hepatocytes. FBPase is a gatekeeper enzyme in the hepatic
gluconeogenesis pathway (glucose is produced from alanine,
glycerol, and lactic acid), which mainly accounts for
endogenous glucose production. To explore whether the in
vivo glucose-lowering effect of W8 resulted from the inhibition
of FBPase, the effects of W8 on gluconeogenesis glucose
output by mouse primary hepatocytes were determined. As
illustrated in Figure 4E, W8 exhibited remarkable dosedependent glucose-lowering effect on mouse primary hepatocytes at concentrations from 50 to 300 μM, and a cellular
viability assay showed that W8 induced no significant toxicity
in hepatocytes (Figure 4F), suggesting that the glucoselowering effect of W8 in hepatocytes was reliable. Furthermore,
we also investigated the glucose-lowering effect of saccharin in
hepatocytes. Saccharin administered at 50 and 100 μM did not
lead to an apparent glucose-lowering effect, while the same
dose of W8 showed an effect that was consistent with our in
vivo results (Figure S12C). These results indicated that the
glucose-lowering effect of W8 resulted from the inhibition of
the hepatic gluconeogenesis pathway.
Pharmacokinetic Properties of Compound W8 and
Saccharin. The concentrations of plasma W8 and its leaving
group saccharin at different times (0−24 h) were determined
(Tables S9 and S10), and the pharmacokinetic properties of
saccharin were evaluated in vivo. It could be seen in Table S9
that a little of W8 was detected within 6 h, but a large amount
of its leaving group saccharin was detected in plasma at 0.25 h
(Table S10), indicating that W8 could react rapidly to generate
saccharin in vivo (Figure S15). Saccharin is further
metabolized in vivo, and its elimination half-live (T1/2), time
to reach maximum plasma concentration (Tmax), maximum
plasma concentration (Cmax), and area under the curve
(AUC(0−t)) were 1.11 h, 0.25 h, 29 922.33 ng/mL, and
14 034.38 h·ng/mL, respectively (Table S11).
■
EXPERIMENTAL SECTION
Chemistry. Common reagents and solvents were purchased from
commercial suppliers and used without further purification unless
otherwise stated. Reaction progress was monitored using analytical
thin-layer chromatography (TLC) on precoated silica gel GF254
plates (QingdaoChem), and spots were detected under UV light (254
and 365 nm). Compounds were purified with flash column
chromatography with silica gel and particle size of 48−74 μM
(Macklin) as the stationary phase and petroleum ether/ethyl acetate
mixture as the eluent system.
1
H and 13C spectra were obtained on a Bruker AV-600 NMR
instrument (Bruker, Karlsruhe, Germany) using deuterated solvents
(DMSO-d6, CDCl3, acetone-d6, D2O), and 19F NMR was recorded
with a Bruker AMX 400 spectrometer with DMSO-d6 and with
tetramethylsilane (TMS) as the internal standard. Chemical shifts are
expressed in ppm relative to DMSO-d6, CDCl3, acetone-d6 or D2O
(2.50/7.26/2.15/4.81 for 1H; 39.52/77.16/29.92 and 206.68 for 13C)
with TMS used as the internal standard. The following abbreviations
for multiplicity were used: s = singlet, d = doublet, t = triplet, m =
multiplet, dd = double doublet, br = broad. High-resolution mass
spectrometry (HRMS) data were obtained by electron ionization (EI)
using a Waters GCT Premier instrument. Compound purity was
determined by high-performance liquid chromatography (HPLC)
chromatograms acquired on a DIONEX UltiMate 3000. Analyses
were performed using a Thermo Fisher Scientific 120 C18 column
(4.6 mm × 250 mm, 5 μm) and acetonitrile for 10 min. Detection was
measured at 254 nm, and the average peak area was used to determine
purity. All of the compounds were determined to be >95% pure.
General Procedure for Synthesizing Compound 2. Saccharin
(0.458 g, 2.5 mmol) was added to a cold solution of lithium
aluminum hydride (0.19 g) in THF (30 mL) that had been
maintained at 0 °C in an external ice bath. The reaction mixture was
allowed to reach ambient temperature and stirred for 1 h. The
reaction was quenched with the addition of water and 2.5 M aqueous
sulfuric acid. The mixture was filtered through Celite and washed with
■
CONCLUSIONS
In this study, the discovery of new saccharin moieties capable
of covalent reactions with cysteine was reported. Exploration of
reactive saccharin derivatives led to the identification of Nacylamino saccharin moieties capable of electrophilic cysteine
capture, as determined by combining 1H NMR, site-directed
mutagenesis, and protein LC−MS, which hold potential
applications as novel chemical biological probes and as new
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2H), 7.35 (t, J = 7.2 Hz, 1H), 5.49 (s, 2H). 13C NMR (151 MHz,
CDCl3): δ 151.13, 142.20, 132.46, 131.55, 130.17, 129.10, 124.04,
123.94, 123.48, 121.50, 120.85, 116.54, 65.05.
N-Benzyl-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8). Compound W8 was synthesized via the method
described by Carnaroglio et al.63 Compound 8 (0.266 g, 2 mmol)
was added to a solution of saccharin (0.366 g, 2 mmol) in ethyl
acetate (30 mL) and refluxed for 1 h. Then, it was cooled and
concentrated in vacuo, the residue was washed with aqueous acetone
solution (50% acetone in water), and the target compound W8 was
obtained as a white powder (0.632 g, 1.5 mmol, 75% yield). 1H NMR
(600 MHz, DMSO-d6): δ 8.66 (s, 1H), 8.32 (s, 1H), 8.19 (d, J = 7.1
Hz, 1H), 8.12 (d, J = 6.5 Hz, 1H), 8.04 (s, 1H), 7.37 (s, 4H), 7.28 (s,
1H), 4.50 (s, 2H). 13C NMR (151 MHz, DMSO-d6): δ 159.28,
148.33, 138.67, 137.56, 137.39, 135.86, 128.83, 127.85, 127.60,
126.33, 124.95, 121.89, 43.56. HRMS (ESI) m/z: calcd for
C15H12N2O4S [M + H]+, 317.0591; found, 317.0583.
3-Oxo-N-(1-phenylethyl)benzo[d]isothiazole-2(3H)-carboxamide
1,1-Dioxide (W9). Compound W9 was synthesized via the same route
as that used for compound W8 as a white powder (0.429 g, 1.30
mmol, 52% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.32 (d, J = 7.2
Hz, 1H), 8.18 (d, J = 7.7 Hz, 2H), 8.13 (d, J = 7.0 Hz, 1H), 8.04 (d, J
= 7.2 Hz, 1H), 7.32 (d, J = 6.7 Hz, 2H), 7.28 (s, 2H), 7.23 (s, 1H),
3.53 (d, J = 3.7 Hz, 2H), 2.87 (s, 2H). 13C NMR (151 MHz, DMSOd6): δ 158.83, 147.64, 138.62, 137.03, 136.91, 135.33, 128.58, 128.39,
126.26, 125.83, 124.27, 121.37, 41.06, 34.69. HRMS (ESI) m/z: calcd
for C16H14N2O4S [M + H]+, 331.0747; found, 331.0741.
3-Oxo-N-propylbenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8a). Compound W8a was synthesized via the same route
as that used for compound W8 as a white powder (0.368 g, 1.38
mmol, 55% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.33 (d, J = 7.7
Hz, 1H), 8.22-8.15 (m, 2H), 8.12 (t, J = 7.5 Hz, 1H), 8.04 (t, J = 7.5
Hz, 1H), 3.25 (dd, J = 12.8, 6.4 Hz, 2H), 1.56 (dd, J = 14.3, 7.1 Hz,
2H), 0.90 (t, J = 7.3 Hz, 3H). 13C NMR (151 MHz, DMSO-d6): δ
159.33, 148.08, 137.55, 137.38, 135.85, 126.29, 124.90, 121.88, 41.73,
22.53, 11.54. HRMS (ESI) m/z: calcd for C11H12N2O4S [M + H]+,
269.0591; found, 269.0585.
3-Oxo-N-(thiophen-2-ylmethyl)benzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8b). Compound W8b was synthesized via
the same route as that used for compound W8 as a white powder
(0.483 g, 1.50 mmol, 60% yield). 1H NMR (600 MHz, DMSO-d6): δ
8.71 (t, J = 5.6 Hz, 1H), 8.33 (d, J = 7.7 Hz, 1H), 8.18 (d, J = 7.6 Hz,
1H), 8.12 (t, J = 7.6 Hz, 1H), 8.03 (t, J = 7.6 Hz, 1H), 7.45 (d, J = 4.6
Hz, 1H), 7.09 (d, J = 2.4 Hz, 1H), 7.00 (dd, J = 4.7, 3.6 Hz, 1H), 4.65
(d, J = 5.9 Hz, 2H). 13C NMR (151 MHz, DMSO-d6): δ 159.20,
148.18, 141.08, 137.52, 137.41, 135.87, 127.18, 126.91, 126.35,
126.08, 124.87, 121.90, 38.55. HRMS (ESI) m/z: calcd for
C13H10N2O4S2 [M + Na]+, 344.9975; found, 344.9974.
N-(Cyclohexylmethyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8c). Compound W8c was synthesized via the
same route as that used for compound W8 as a white powder (0.483
g, 1.50 mmol, 60% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.30 (s,
1H), 8.16 (d, J = 5.5 Hz, 1H), 8.11 (s, 2H), 8.02 (s, 1H), 3.12 (s,
2H), 1.68 (s, 4H), 1.61 (s, 1H), 1.52 (s, 1H), 1.24-1.08 (m, 3H), 0.93
(d, J = 10.6 Hz, 2H). 13C NMR (151 MHz, DMSO-d6): δ 159.43,
148.09, 137.55, 137.37, 135.83, 126.27, 124.92, 121.86, 45.98, 37.62,
30.49, 26.33, 25.72. HRMS (ESI) m/z: calcd for C15H18N2O4S [M +
Na]+, 345.0879; found, 345.0874.
N-(Naphthalen-1-ylmethyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8d). Compound W8d was synthesized via
the same route as that used for compound W8 as a white powder
(0.458 g, 1.25 mmol, 50% yield). 1H NMR (600 MHz, DMSO-d6): δ
8.71 (t, J = 5.5 Hz, 1H), 8.32 (d, J = 7.7 Hz, 1H), 8.20 (d, J = 8.3 Hz,
1H), 8.17 (d, J = 7.6 Hz, 1H), 8.11 (t, J = 7.6 Hz, 1H), 8.03 (t, J = 7.6
Hz, 1H), 7.97 (t, J = 10.2 Hz, 1H), 7.89 (d, J = 8.1 Hz, 1H), 7.59
(ddd, J = 22.2, 15.0, 7.1 Hz, 3H), 7.51 (t, J = 7.6 Hz, 1H), 4.98 (d, J =
5.7 Hz, 2H). 13C NMR (151 MHz, DMSO-d6): δ 159.37, 148.28,
137.53, 137.39, 135.86, 133.73, 131.06, 129.04, 128.33, 126.90,
126.35, 125.89, 124.96, 123.72, 121.90, 41.62. HRMS (ESI) m/z:
calcd for C19H14N2O4S [M + Na]+, 389.0566; found, 389.0556.
ethyl acetate. The organic layer was washed with 1 M aqueous sulfuric
acid, dried (with anhydrous magnesium sulfate), filtered, and
concentrated to give target compound 2, which was a pale-yellow
powder (0.36 g, 2.125 mmol, 85% yield). 1H NMR (600 MHz,
DMSO-d6): δ 7.81 (d, J = 6.7 Hz, 2H), 7.69 (t, J = 7.2 Hz, 1H), 7.57
(s, 2H), 4.42 (s, 2H). 13C NMR (151 MHz, DMSO-d6): δ 138.29,
136.53, 133.13, 129.37, 125.80, 120.96, 45.33.
General Procedure for Synthesizing (1,1-Dioxidobenzo[d]isothiazol-2(3H)-yl)(phenyl)methanone (W2). A solution of benzoyl
chloride (0.281 g, 2 mmol) in DCM was added dropwise to a stirred
solution of compound 2 (0.338 g, 2 mmol) in DCM with Et3N (0.202
g, 2 mmol), and the solution was stirred at room temperature for 1.5
h. The reaction solution was filtered and concentrated in vacuo, and
the residue was recrystallized with dichloromethane to give target
compound W2, which was a white powder (0.410 g, 1.50 mmol, 30%
yield). 1H NMR (600 MHz, CDCl3): δ 7.87 (d, J = 7.7 Hz, 2H), 7.76
(d, J = 7.8 Hz, 1H), 7.70 (t, J = 7.6 Hz, 1H), 7.62−7.55 (m, 2H), 7.51
(t, J = 7.3 Hz, 3H), 5.20 (s, 2H). 13C NMR (151 MHz, CDCl3): δ
168.72, 134.47, 134.05, 133.94, 132.40, 131.12, 129.62, 128.34,
128.30, 124.83, 121.79, 47.80. HRMS (ESI) m/z: calcd for
C14H11NO3S [M + H]+, 274.0532; found, 274.0529.
2-Benzoylisoindolin-1-one (W3). Compound W3 was synthesized
via the same route as that used for compound W2 as a faint white
powder (0.245 g, 0.9 mmol, 45% yield). 1H NMR (600 MHz,
CDCl3): δ 7.87 (d, J = 7.4 Hz, 1H), 7.70 (d, J = 6.8 Hz, 3H), 7.56 (t, J
= 8.5 Hz, 2H), 7.52 (t, J = 7.2 Hz, 1H), 7.46 (t, J = 7.2 Hz, 2H), 5.06
(s, 2H). 13C NMR (151 MHz, CDCl3): δ 170.43, 166.87, 141.35,
134.39, 134.17, 131.85, 131.03, 128.75, 128.70, 127.78, 125.34,
123.43, 48.82.
2-(2-Phenylacetyl)benzo[d]isothiazol-3(2H)-one 1,1-Dioxide
(W4). A solution of benzoyl chloride (0.281 g, 2 mmol) in ethyl
acetate was added dropwise to a stirred solution of saccharin (0.366 g,
2 mmol) in ethyl acetate (20 mL) with Et3N (0.202 g, 2 mmol), and
the solution was stirred at room temperature for 2 h. The reaction
solution was filtered and concentrated in vacuo, and the residue was
recrystallized from dichloromethane to give target compound W4,
which was a white powder (0.499 g, 1.66 mmol, 83% yield). 1H NMR
(600 MHz, CDCl3): δ 8.14 (d, J = 7.7 Hz, 1H), 7.96 (q, J = 7.4 Hz,
2H), 7.90 (t, J = 7.1 Hz, 1H), 7.34 (d, J = 4.3 Hz, 4H), 7.29 (dd, J =
8.7, 4.3 Hz, 1H), 4.38 (s, 2H). 13C NMR (151 MHz, CDCl3): δ
164.26, 152.63, 133.43, 131.73, 130.13, 126.76, 124.90, 123.92,
122.83, 121.57, 120.15, 116.44, 39.32. HRMS (ESI) m/z: calcd for
C15H11NO4S [M + Na]+, 324.0301; found, 324.0295.
2-(3-Phenylpropanoyl)benzo[d]isothiazol-3(2H)-one 1,1-Dioxide
(W5). Compound W5 was synthesized via the same route as that used
for compound W4 as a white powder (0.511 g, 1.62 mmol, 81%
yield). 1H NMR (600 MHz, CDCl3): δ 8.13 (d, J = 7.6 Hz, 1H),
8.02−7.93 (m, 2H), 7.93−7.87 (m, 1H), 7.35−7.24 (m, 4H), 7.22 (t,
J = 7.0 Hz, 1H), 3.36 (t, J = 7.6 Hz, 2H), 3.08 (t, J = 7.6 Hz, 2H). 13C
NMR (151 MHz, CDCl3): δ 165.78, 152.82, 134.98, 133.36, 131.76,
130.19, 123.84, 123.81, 121.70, 121.52, 120.19, 116.47, 35.13, 24.73.
3-Oxobenzo[d]isothiazole-2(3H)-carboxylate 1,1-Dioxide (W6).
Compound W6 was synthesized via the same route as that used for
compound W4 as a white powder (0.533 g, 1.76 mmol, 88% yield).
1
H NMR (600 MHz, CDCl3): δ 8.22 (d, J = 7.5 Hz, 1H), 8.02 (s,
2H), 7.95 (d, J = 5.0 Hz, 1H), 7.45 (d, J = 7.4 Hz, 2H), 7.40−7.30
(m, 3H). 13C NMR (151 MHz, CDCl3): δ 155.92, 149.48, 145.35,
137.24, 136.47, 135.04, 129.66, 126.99, 126.40, 125.57, 121.44,
121.16. HRMS (ESI) m/z: calcd for C14H9NO5S [M + H]+,
304.0274; found, 304.0273.
Benzyl 3-Oxobenzo[d]isothiazole-2(3H)-carboxylate 1,1-Dioxide
(W7). Benzyloxycarbonyl chloride (0.853 g, 5 mmol) was added
dropwise to a suspension of saccharin sodium salt (1.025 g, 5 mmol)
in ice-cold tetrahydrofuran (50 mL) with stirring. The mixture was
maintained at 50 °C for 6 h and at room temperature for an additional
12 h. The insoluble material was removed by filteration, and the
filtrate was evaporated. The residue was crystallized from THF to give
W7 as a white solid (1.443 g, 4.55 mmol, 91% yield). 1H NMR (600
MHz, CDCl3): δ 8.15 (d, J = 7.7 Hz, 1H), 7.95 (s, 2H), 7.89 (ddd, J =
8.1, 5.4, 3.0 Hz, 1H), 7.50 (d, J = 7.3 Hz, 2H), 7.39 (t, J = 7.3 Hz,
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3-Oxo-N-(1-phenylethyl)benzo[d]isothiazole-2(3H)-carboxamide
1,1-Dioxide (W8e). Compound W8e was synthesized via the same
route as that used for compound W8 as a white powder (0.511 g, 1.55
mmol, 62% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.42 (d, J = 6.8
Hz, 1H), 8.32 (d, J = 7.5 Hz, 1H), 8.19 (d, J = 7.3 Hz, 1H), 8.12 (t, J
= 7.2 Hz, 1H), 8.05 (d, J = 7.4 Hz, 1H), 7.44 (d, J = 7.0 Hz, 2H), 7.38
(s, 2H), 7.30 (d, J = 6.4 Hz, 1H), 5.08−4.94 (m, 1H), 1.53 (d, J = 6.5
Hz, 3H). 13C NMR (151 MHz, DMSO-d6): δ 159.53, 147.27, 143.35,
137.49, 137.44, 135.93, 128.98, 127.73, 126.48, 126.32, 125.00,
121.94, 50.20, 22.67. HRMS (ESI) m/z: calcd for C16H14N2O4S [M +
Na]+, 353.0566; found, 353.0565.
N-(2-Fluorobenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8f). Compound W8f was synthesized via the
same route as that used for compound W8 as a white powder (0.400
g, 1.20 mmol, 48% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.66 (s,
1H), 8.34 (d, J = 7.5 Hz, 1H), 8.21 (d, J = 7.5 Hz, 1H), 8.14 (t, J = 7.2
Hz, 1H), 8.06 (d, J = 7.5 Hz, 1H), 7.48 (d, J = 7.1 Hz, 1H), 7.37 (s,
1H), 7.24 (d, J = 13.7 Hz, 2H), 4.57 (d, J = 5.1 Hz, 2H). 13C NMR
(151 MHz, DMSO-d6): δ 161.27, 159.49 (d, J = 48.9 Hz), 148.35,
137.53, 137.42, 135.88, 129.94, 129.78, 126.34, 125.37 (d, J = 13.9
Hz), 124.87, 121.91, 115.69, 115.55, 37.66. HRMS (ESI) m/z: calcd
for C15H11N2O4FS [M + H]+, 335.0496; found, 335.0496.
N-(2-Chlorobenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8g). Compound W8g was synthesized via the
same route as that used for compound W8 as a white powder (0.420
g, 1.20 mmol, 48% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.68 (d,
J = 5.4 Hz, 1H), 8.31 (d, J = 7.7 Hz, 1H), 8.18 (d, J = 7.6 Hz, 1H),
8.10 (t, J = 7.6 Hz, 1H), 8.02 (t, J = 7.6 Hz, 1H), 7.50−7.41 (m, 2H),
7.39−7.27 (m, 2H), 4.55 (d, J = 5.9 Hz, 2H). 13C NMR (151 MHz,
DMSO-d6): δ 159.38, 148.42, 137.53, 137.44, 135.91, 135.65, 132.43,
129.67, 129.48, 129.35, 127.76, 126.37, 124.98, 121.94, 41.59. HRMS
(ESI) m/z: calcd for C15H11ClN2O4S [M + H]+, 351.0201; found,
351.0200.
N-(2-Bromobenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8h). Compound W8h was synthesized via the
same route as that used for compound W8 as a white powder (0.583
g, 1.40 mmol, 56% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.70 (s,
1H), 8.31 (s, 1H), 8.18 (s, 1H), 8.10 (s, 1H), 8.02 (s, 1H), 7.63 (s,
1H), 7.44 (s, 1H), 7.38 (s, 1H), 7.23 (s, 1H), 4.52 (s, 2H). 13C NMR
(151 MHz, DMSO-d6): δ 159.38, 148.42, 137.51, 137.42, 137.16,
135.89, 132.89, 129.73, 129.34, 128.29, 126.35, 124.96, 122.68,
121.92, 43.99. HRMS (ESI) m/z: calcd for C15H11BrN2O4S [M +
Na]+, 416.9515; found, 416.9517.
3-Oxo-N-(2-(trifluoromethyl)benzyl)benzo[d]isothiazole-2(3H)carboxamide 1,1-Dioxide (W8i). Compound W8i was synthesized
via the same route as that used for compound W8 as a white powder
(0.424 g, 1.13 mmol, 45% yield). 1H NMR (600 MHz, DMSO-d6): δ
8.73 (d, J = 5.3 Hz, 1H), 8.31 (d, J = 7.7 Hz, 1H), 8.18 (d, J = 7.6 Hz,
1H), 8.10 (t, J = 7.6 Hz, 1H), 8.02 (t, J = 7.5 Hz, 1H), 7.73 (d, J = 7.7
Hz, 1H), 7.67 (t, J = 7.5 Hz, 1H), 7.62 (d, J = 7.6 Hz, 1H), 7.49 (t, J =
7.4 Hz, 1H), 4.66 (d, J = 5.5 Hz, 2H). 13C NMR (151 MHz, DMSOd6): δ 159.35, 148.50, 137.51, 137.46, 136.80, 135.92, 133.27, 129.01,
128.17, 126.61, 126.37, 125.75, 124.95, 123.93, 121.95, 120.92. 19F
NMR (376 MHz, DMSO-d6): δ −58.98 (s). HRMS (ESI) m/z: calcd
for C16H14N2O4F3S [M + Na]+, 407.0284; found, 407.0279.
N-(2-Methylbenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8j). Compound W8j was synthesized via the
same route as that used for compound W8 as a white powder (0.454
g, 1.28 mmol, 55% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.54 (s,
1H), 8.33 (d, J = 7.7 Hz, 1H), 8.19 (d, J = 7.6 Hz, 1H), 8.12 (t, J = 7.6
Hz, 1H), 8.04 (t, J = 7.6 Hz, 1H), 7.33 (d, J = 3.8 Hz, 1H), 7.20 (s,
3H), 4.49 (d, J = 5.7 Hz, 2H), 2.34 (s, 3H). 13C NMR (151 MHz,
DMSO-d6): δ 159.37, 148.16, 137.54, 137.40, 136.24, 136.04, 135.89,
135.09, 130.50, 127.92, 127.70, 126.33, 125.01, 121.92, 41.70, 19.10.
HRMS (ESI) m/z: calcd for C16H14N2O4S [M + Na]+, 353.0566;
found, 353.0559.
N-(2-Methoxybenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8k). Compound W8k was synthesized via the
same route as that used for compound W8 as a white powder (0.346
g, 1.00 mmol, 40% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.52 (s,
Article
1H), 8.29 (d, J = 7.6 Hz, 1H), 8.16 (d, J = 7.6 Hz, 1H), 8.09 (t, J = 7.5
Hz, 1H), 8.00 (t, J = 7.5 Hz, 1H), 7.27 (t, J = 8.3 Hz, 2H), 7.01 (d, J =
7.9 Hz, 1H), 6.91 (t, J = 7.2 Hz, 1H), 4.43 (d, J = 5.5 Hz, 2H), 3.83
(s, 3H). 13C NMR (151 MHz, DMSO-d6): δ 159.58, 157.31, 148.22,
137.51, 137.39, 135.86, 129.26, 128.68, 126.36, 125.72, 124.97,
121.89, 120.71, 111.14, 55.88, 39.46. HRMS (ESI) m/z: calcd for
C16H14N2O5S [M + Na]+, 369.0516; found, 369.0518.
N-(3-Bromobenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8l). Compound W8l was synthesized via the
same route as that used for compound W8 as a white powder (0.581
g, 1.48 mmol, 59% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.71 (s,
1H), 8.29 (d, J = 6.2 Hz, 1H), 8.16 (d, J = 6.0 Hz, 1H), 8.08 (s, 1H),
8.00 (s, 1H), 7.56 (s, 1H), 7.44 (s, 1H), 7.34 (s, 1H), 7.29 (s, 1H),
4.45 (s, 2H). 13C NMR (151 MHz, DMSO-d6): δ 159.21, 148.44,
141.67, 137.54, 137.37, 135.87, 131.00, 130.58, 130.43, 126.99,
126.34, 125.02, 122.09, 121.90, 43.01. HRMS (ESI) m/z: calcd for
C15H11BrN2O4S [M + H]+, 394.9696; found, 394.9693.
N-(3-Methoxybenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8m). Compound W8m was synthesized via the
same route as that used for compound W8 as a white powder (0.652
g, 1.75 mmol, 70% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.66 (s,
1H), 8.33 (d, J = 7.7 Hz, 1H), 8.19 (d, J = 7.5 Hz, 1H), 8.12 (t, J = 7.4
Hz, 1H), 8.04 (t, J = 7.5 Hz, 1H), 7.28 (t, J = 7.7 Hz, 1H), 6.95 (d, J =
7.9 Hz, 2H), 6.85 (d, J = 7.8 Hz, 1H), 4.47 (d, J = 5.5 Hz, 2H), 3.75
(s, 3H). 13C NMR (151 MHz, DMSO-d6): δ 159.75, 159.25, 148.28,
140.20, 137.55, 137.37, 135.84, 129.91, 126.31, 124.96, 121.88,
119.91, 113.58, 112.82, 55.41, 43.49. HRMS (ESI) m/z: calcd for
C16H14N2O5S [M + Na]+, 396.0516; found, 396.0510.
3-Oxo-N-(4-(trifluoromethyl)benzyl)benzo[d]isothiazole-2(3H)carboxamide 1,1-Dioxide (W8n). Compound W8n was synthesized
via the same route as that used for compound W8 as a white powder
(0.610 g, 1.58 mmol, 63% yield). 1H NMR (600 MHz, DMSO-d6): δ
8.81 (s, 1H), 8.33 (d, J = 7.3 Hz, 1H), 8.21 (d, J = 7.3 Hz, 1H), 8.13
(t, J = 7.3 Hz, 1H), 8.05 (t, J = 6.8 Hz, 1H), 7.73 (d, J = 7.4 Hz, 2H),
7.60 (d, J = 7.0 Hz, 2H), 4.58 (d, J = 5.4 Hz, 2H). 13C NMR (151
MHz, DMSO-d6): δ 159.21, 148.50, 143.71, 137.55, 137.41, 135.88,
128.48, 128.07 (t, J = 114 Hz), 127.95, 126.33, 125.68, 124.93,
121.91, 43.23. 19F NMR (376 MHz, DMSO-d6): δ −60.83 (s).
HRMS (ESI) m/z: calcd for C16H11F3N2O4S [M + H]+, 385.0464;
found, 385.0467.
N-(4-Bromobenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8o). Compound W8o was synthesized via the
same route as that used for compound W8 as a white powder (0.604
g, 1.53 mmol, 61% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.69 (s,
1H), 8.28 (s, 1H), 8.15 (s, 1H), 8.09 (s, 1H), 8.00 (s, 1H), 7.52 (s,
2H), 7.30 (s, 2H), 4.42 (s, 2H). 13C NMR (151 MHz, DMSO-d6): δ
159.22, 148.40, 138.28, 137.54, 137.41, 135.88, 131.68, 130.14,
126.34, 124.93, 121.91, 120.65, 43.01. HRMS (ESI) m/z: calcd for
C15H11BrN2O4S [M + H]+, 394.9695; found, 394.9696.
N-(4-Methoxybenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8p). Compound W8p was synthesized via the
same route as that used for compound W8 as a white powder (0.520
g, 1.5 mmol, 60% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.56 (s,
1H), 8.29 (d, J = 7.6 Hz, 1H), 8.15 (d, J = 7.6 Hz, 1H), 8.09 (t, J = 7.5
Hz, 1H), 8.00 (t, J = 7.5 Hz, 1H), 7.28 (d, J = 8.2 Hz, 2H), 6.89 (d, J
= 8.3 Hz, 2H), 4.38 (d, J = 5.5 Hz, 2H), 3.71 (s, 3H). 13C NMR (151
MHz, DMSO-d6): δ 159.29, 158.92, 148.20, 137.55, 137.39, 135.86,
130.57, 129.43, 126.32, 124.93, 121.89, 114.22, 55.50, 43.07. HRMS
(ESI) m/z: calcd for C16H14N2O5S [M + Na]+, 369.0510; found,
369.0516.
N-(4-Methylbenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (W8q). Compound W8q was synthesized via the
same route as that used for compound W8 as a white powder (0.538
g, 1.63 mmol, 65% yield). 1H NMR (600 MHz, DMSO-d6): δ 8.62 (s,
1H), 8.32 (d, J = 7.2 Hz, 1H), 8.18 (d, J = 7.1 Hz, 1H), 8.11 (d, J =
6.9 Hz, 1H), 8.04 (d, J = 7.0 Hz, 1H), 7.27 (d, J = 6.7 Hz, 2H), 7.17
(d, J = 6.7 Hz, 2H), 4.44 (d, J = 4.3 Hz, 2H), 2.29 (s, 3H). 13C NMR
(151 MHz, DMSO-d6): δ 159.29, 148.25, 137.55, 137.40, 136.75,
135.87, 135.61, 129.38, 127.90, 126.33, 124.94, 121.90, 43.34, 21.14.
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HRMS (ESI) m/z: calcd for C16H14N2O4S [M + H]+, 331.0747;
found, 331.0743.
N-(4-Ethynylbenzyl)-3-oxobenzo[d]isothiazole-2(3H)-carboxamide 1,1-Dioxide (AP1). tert-Butyl (4-ethynylbenzyl)carbamate (9,
0.462 g, 2 mmol) was resolved in DCM (20 mL), then CF3COOH (2
mL) was added, and the solution was stirred at 0 °C for 12 h. Then, it
was concentrated in vacuo, the residue was resolved in saturated
sodium carbonate aqueous solution and extracted with ethyl acetate
(3 × 100 mL). The organic layer was dried with anhydrous
magnesium sulfate, filtered, and concentrated to give compound (4ethynylphenyl)methanamine (10). Then, compound AP1 was
synthesized via the same route as that used for compound W8 as a
white powder (0.306 g, 0.9 mmol, 45% yield). 1H NMR (600 MHz,
DMSO-d6): δ 8.73 (s, 1H), 8.33 (d, J = 7.3 Hz, 1H), 8.20 (d, J = 7.0
Hz, 1H), 8.12 (d, J = 7.1 Hz, 1H), 8.05 (d, J = 7.2 Hz, 1H), 7.48 (d, J
= 7.3 Hz, 2H), 7.39 (d, J = 7.3 Hz, 2H), 4.51 (d, J = 4.7 Hz, 2H), 4.18
(s, 1H). 13C NMR (151 MHz, DMSO-d6): δ 159.23, 148.41, 139.76,
137.54, 137.39, 135.86, 132.16, 128.06, 126.32, 124.93, 121.89,
120.89, 83.77, 81.17, 43.31. HRMS (ESI) m/z: calcd for
C17H12N2O4S [M + Na]+, 363.0415; found, 363.0415.
NMR Method to Determine Kinetic Parameters. The analysis
method from reported procedures was adopted with modifications.64,65 The reaction temperature was 37 °C in all cases. A 500
μL aliquot of the 40 mM test compound was placed in an NMR tube
with 5 mm outside diameter (OD). A freshly prepared solution (100
μL) of nucleophile stock (30 μmol) in buffer (300 mM, pH 7.4, D2O)
was transferred to the NMR tube that contained reagents and was
quickly inverted several times to aid mixing and dissolution; thus, the
reaction was initiated. The thoroughly mixed solution was inserted
into the NMR machine cavity, and the acquisition of 1H NMR data
was immediately initiated (TMS was used as an internal standard for
the NMR analysis as it did not interfere with the analyses). The
pseudo-first-order rate constants were determined by plotting the
natural log of the electrophile/internal standard ratio as a function of
time, as defined by the area of a given resonance OD an electrophile
or internal standard vs time. The negative slope of the straight line is
the pseudo-first-order rate constant.
inhibition assays, mass spectrometry studies, protein crystallization,
etc.
In Vitro Biological assays. For the human liver FBPase
inhibition assay, the enzymatic activities of Hu-FBPase were measured
by a colorimetric assay based on the detection of inorganic phosphate
hydrolyzed from FBP, as described in our previous work.67 The
released phosphate was quantified in a complex with ammonium
molybdate and malachite green by spectrophotometry. To calculate
the product formation at the micromolar level, calibration curves were
generated using a standard KH2PO4 solution in the range of 2−60
μM. The absorbance of the reaction mixture was measured at 620 nm
with a spectrophotometer (SpectraMax M5, Molecular Devices).
To determine the corresponding inhibitor constants (IC50 values),
initial rate data of the saturating substrate, fixed effector, and
systematically varied inhibitor concentrations were fit to the Hill
equation: V = V0 − (V0 − V∞)/[(IC50/I)n + 1], where V, V0, and V∞
are the velocity, maximum velocity (at I = 0), and limiting velocity (at
I saturation), respectively; n is the Hill coefficient associated with the
inhibitor; and IC50 is the concentration of inhibitor to reach a 50%
inhibition rate. All kinetic data were fit to a growth/sigmoidal model
with Origin 7.5 software.
Selectivity Evaluation of Some Saccharin Derivatives. The
pET28a expression vector containing the human GAPDH gene was
cloned. The enzyme was expressed in E. coli BL21 (DE3) and purified
to homogeneity as previously described.68 In vitro recombinant
human GAPDH activity was measured by spectrophotometry as
described by Kornberg with slight modifications.69 Assays were
performed with 10 mM sodium pyrophosphate buffer (pH 8.5) in 96well plates. First, 495 μL of recombinant GAPDH (0.1 mg/mL final
concentration) was incubated with 5 μL of test compounds for 30
min. Then, the enzymatic activity was measured with a microplate
reader spectrophotometer (SpectraMax M5, Molecular Devices), with
absorbance at 340 nm, indicating the reduction of NAD+. The assay
was performed at 37 °C. An additional 200 μL of reaction mixture
containing sodium arsenate, 4 mM NAD+, and 12 mM glyceraldehyde
3-phosphate (G3P) was then rapidly added to each well to start the
reaction. The absorbance was measured at 340 nm 4 min after
reaction initiation.
A nicotinamide adenine dinucleotide (NADH)-linked enzymatic
assay was performed to measure the inhibitory activity of compounds
against aldolase.70 Commercial preparations of glycerol 3-phosphate
dehydrogenase (GPDH) from rabbit muscle and triosephosphate
isomerase (TPI) from rabbit muscle, both obtained from Sigma, were
used. Recombinant aldolase (ALDOA/ALDOB/ALDOC) was mixed
with serial dilutions of each compound, NADH (0.41 mM) and
triosephosphate isomerase (TPI) (0.0025 U/μL, Sigma), in 412 μL of
assay buffer (0.1 M Tris, pH 7.4 and 0.2 M potassium-acetate) and
incubated for 3 min at 37 °C. The reaction was initiated by adding 4.2
μL of FBP (100 mM) and 4.2 μL of GAPDH (0.0625 U/μL, Sigma).
The decrease in NADH absorbance at 340 nm was measured every 30
s for 6 min with a spectrophotometer (SpectraMax M5, Molecular
Devices). Initial velocities of reactions with the same compounds used
in combination with DMSO were calculated and used to generate
IC50 curves.
The kinase assays were carried out as described previously.71 All of
the enzymatic reactions were conducted at 30 °C for 40 min. The 50
μL reaction mixture contains 40 mM Tris, pH 7.4, 10 mM MgCl2, 0.1
mg/mL bovine serum albumin (BSA), 1 mM dithiothreitol (DTT),
10 μM adenosine 5′-triphosphate (ATP), kinase, and the substrate.
The compounds were diluted in 10% DMSO, and 5 μL of the dilution
was added to a 50 μL reaction so that the final concentration of
DMSO is 1% in all of the reactions. The assay was performed using
the Kinase-Glo Plus luminescence kinase assay kit. It measures kinase
activity by quantitating the amount of ATP remaining in the solution
following a kinase reaction. The luminescent signal from the assay is
correlated with the amount of ATP present and is inversely correlated
with the amount of kinase activity.
Preparation of Cell Lysates. Hepatic LO2 cells were maintained
in Roswell Park Memorial Institute (RPMI)-1640 medium (Procell)
supplemented with 10% fetal bovine serum (FBS) and 1% antibiotics.
ln([electrophile]) = − k pseudo 1st × t + ln([electrophile0])
t1/2 =
Article
0.693
(60×k pseudo 1st)
Protein Expression and Purification. The cDNA of Hu-FBPase
(GenBank: D26055.1) was cloned into an EX-C0133-B01 vector. To
obtain the purified protein, an eight amino acid sequence
(SYYHHHHHH) was added to the N-terminus of Hu-FBPase. The
plasmid was then transformed into BL21 (DE3) cells for protein
expression and purified with a HisTrap_FF_5 mL [Global] column
with the standard Ä KTA pure system, as described in our previous
work.66 The process for purifying hGAPDH was the same as that used
to purify Hu-FBPase, as described above. The cDNA of ALDOA
(GenBank: CR541880.1), ALDOB (GenBank: KR711267.1), and
ALDOC (GenBank: CR541881.1) was cloned into an pET28a vector.
The transformation and purification processes applied to the aldolases
were the same as those used for Hu-FBPase, as described above.
Site-directed mutagenesis experiments were performed by
introducing specific bases into a double-stranded DNA plasmid.
Mutant constructs were generated using the two-step polymerase
chain reaction (PCR) method. DNA encoding WT Hu-FBPase was
cloned into EX-C0133-B01 and used as the template for mutagenesis.
Parental methylated and hemimethylated DNA were digested by the
NspV and NotI restriction enzymes. Then, the mutant constructs were
ligated into a previous plasmid. The plasmids carrying the
recombinant mutant were transformed into DH5α competent cells.
All of the mutations were confirmed by DNA sequencing. The verified
plasmids with mutations were transformed into the Escherichia coli
BL21 (DE3) strain cells. The mutant Hu-FBPase proteins were
purified in the same manner as WT Hu-FBPase. The eluted protein
samples were stored in a solution (10 mM Tris, pH 7.5) for enzymatic
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The cell lines were grown at 37 °C in a 5% CO2 atmosphere. LO2
cells were grown in the culture medium until 70−80% confluence,
then the medium was removed, and the cells were washed twice with
cold PBS (10 mL) and lysed with 200 μL of cell lysis buffer for
western blot and IP (containing protease inhibitor, Beyotime)
analyses. The lysed cells were centrifuged (15 000g, 10 min) at 4
°C. The supernatant was transferred to a separated microfuge tube
and stored at −20 °C. After thawing the supernatant on ice, the
protein concentration was determined using the bicinchoninic acid
(BCA) protein assay kit (Beyotime) and adjusted to 2 mg/mL by
dilution with PBS.
In Vitro Labeling of LO2 Cell Lysate Proteomes. The cell
lysate (48 μL containing 2 mg/mL proteins) was treated with a series
of concentrations of AP1 (1 μL) at 37 °C for 2 h. The solution was
subjected to a copper-catalyzed azide−alkyne cycloaddition (CuAAC)
reaction with 0.5 mM biotin-PEG3-azide, 0.5 mM sodium ascorbate,
0.5 mM 3,3′,3″-[nitrilotris(methylene-1H-1,2,3-triazole-4,1-diyl)] tri(1-propanol), and 0.5 mM CuSO4. The mixture was incubated at 37
°C for 1 h. After CC, 50 μL of 2× sodium dodecyl sulfate (SDS)
loading buffer was added to the mixture to stop the reaction, which
was further heated at 95 °C for 10 min. The solution was used for
subsequent silver staining and streptavidin blotting.
Streptavidin Blot Analysis. Denatured protein samples were
resolved by 10% SDS-polyacrylamide gel electrophoresis (PAGE),
and then, the separated proteins were transferred to poly(vinylidene
difluoride) (PVDF) membranes, which were washed with Trisbuffered saline containing 0.1% Tween 20 (TBST) three times
powdered milk for 1.5 h, incubated with streptavidin−horseradish
peroxidase (HRP, Beyotime, with 1:5000 dilution in 5% nonfat dried
milk TBST solution) overnight at 4 °C for biotin labels on blots.
Finally, the membrane was washed with PBST, and signals were
detected by ECL western blot detection reagents (Beyotime) using a
ChemiDoc XRS+ system (Bio-Rad).
Protein Crystallization and Structure Determination. W8
was added to the protein such that the final inhibitor concentration
after the subsequent addition of the sample was 10 mM. The sample
was concentrated by ultrafiltration to a protein concentration of 8
mg/mL (measured by absorbance at 280 nm). Crystals were obtained
using a hanging-drop vapor diffusion method at 18 °C. Crystals grew
from a mixture of 1 μL of protein and 1 μL of a well solution
containing 1 mM adenosine monophosphate (AMP), 0.1 M Tris (pH
= 6.0) and 14% v/v EtOH. Crystals were cryoprotected using a well
solution supplemented with 25% glycerol and flash-frozen in liquid
nitrogen. X-ray diffraction data were collected at the BL19U1
beamline at the Shanghai Synchrotron Radiation Facility. The
statistics related to data are presented in Table S6.
The diffraction data were indexed, integrated, and scaled using
XDS.72 The structure was resolved by molecular replacement using
Phaser,73 and the structure of Hu-FBPase (PDB ID: 5ZWK) was used
as the search model.74 There were four molecules in the asymmetric
unit. The model was built using Coot70 and refined with PHENIX.75
Atomic restraints were generated for the inhibitor using eLBOW,76
and the model was validated using MolProbity.77
Mass Spectrometry. For precipitation and digestion, proteins
were precipitated with precooled acetone. The protein pellet was
dried by a SpeedVac for 1−2 min. The pellet was subsequently
dissolved in 8 M urea and then diluted ten times with 100 mM Tris−
HCl. The protein source was digested overnight with Glu-C at a 1:20
ratio (w/w) (Promega, https://www.promega.com.cn/). The reaction
was stopped by adding formic acid, and the peptide solution was
desalted with a Monospin C18 column (Shimadzu GL).
For the LC-tandem MS (MS/MS) analyses of peptides, the peptide
mixture was solubilized in 0.1% formic acid and loaded onto a
homemade 30 cm long pulled-tip analytical column (ReproSil-Pur
C18 AQ with a 1.9 μm particle size, Dr. Maisch GmbH, 75 μm ID ×
360 μm OD) connected to an Easy-nLC1200 UHPLC (Thermo
Scientific) for mass spectrometry analysis (Q Exactive Orbitrap mass
spectrometer, Thermo Scientific, San Jose, CA). The elution gradients
and mobile phases used for peptide separations are constituted as
follows: for 0−1 min, 3−6% B; for 1−96 min, 6−30% B; for 96−114
Article
min, 30−60% B; for 114−115 min, 60−100% B; for 115−120 min,
and for 100−100% B (mobile phase A, 0.1% formic acid in water and
mobile phase, phase B, 0.1% formic acid in 80% acetonitrile) at a flow
rate of 300 nL/min. Peptides eluted from the LC column were
directly electrosprayed into the mass spectrometer with the
application of a distal 1.8 kV spray voltage. Survey full-scan MS
spectra (from m/z 300 to 1800) were acquired with an Orbitrap
analyzer (Q Exactive) at a resolution of r = 70 000 at m/z 400. The
top 20 MS/MS events were sequentially generated from the full MS
spectrum at a 30% normalized collision energy. The dynamic
exclusion time was 10 s.
Data Analysis. The acquired MS/MS data were analyzed against a
database downloaded from UniProt using PEAKS (version 8.5). To
estimate peptide probabilities and false discovery rates accurately, we
used a decoy database containing the reversed sequences of all of the
proteins and appended it to the target database. Mass tolerances for
the precursor ions were set at 20 ppm, and for MS/MS, they were set
at 0.02 Da. The specific binding was set as a dynamic modification
with a mass shift of 342.00198 at cysteine.
Glucose Production Assay. Primary mouse hepatocytes treated
overnight with serum starvation were treated with compounds in a
DMEM environment for 6 h, and the effect of metformin (1 mM),
saccharin (50 or 100 μM), and the test compounds (50−300 μM) on
the level of gluconeogenesis in primary hepatocytes was measured by
assaying the level of glucose in their culture medium.
Animals. Male ICR mice (20−24 g) were obtained from Shanghai
JSJ Lab Animal, Ltd. The mice were cultured under a specific
pathogen-free environment with a 12 h light−dark cycle, relative
humidity of 55−60%, a temperature of 22−24 °C, and free access to
water and food. The animal studies were approved by the Animal
Care and Use Committee of Central China Normal University
(CCNU-IACUC-2021-006).
Glucose Reduction in ICR Mice. ICR mice (n = 4 in each group)
fasted for 12 h were intraperitoneally administered vehicle (10%
ricinus oil in water), metformin (250 mg/kg), or a test compound (30
mg/kg). Food was withheld throughout the study. Blood samples
were collected from the tail vein 0, 1, 2, 4, and 6 h after treatment and
analyzed by a glucometer (Sanicare).
OGTT Was Performed with ICR Mice. ICR mice that had been
fasted for 12 h (n = 6 in each group) were intraperitoneally
administered compound W8 (10 and 30 mg/kg), metformin (250
mg/kg), or vehicle (10% ricinus oil in water) 1 h before the glucose
challenge. Glucose (2 g/kg) was orally administered at 0 h, and blood
samples were drawn from the tail vein 0, 0.25, 0.5, 1, 1.5, and 2 h after
glucose administration. Plasma glucose was measured using a
glucometer (Sanicare). Food was withheld throughout the study.
Pharmacokinetic Studies. The pharmacokinetic studies of W8
and its leaving group saccharin were performed by Shanghai
Medicilon Inc. ICR mice were fasted for 12 h before intraperitoneal
administration of 30 mg/kg W8. Water and food were allowed free
access after 4 h of intraperitoneal administration. The blood of ICR
mice was collected at different time points (0.25, 0.5, 1, 2, 4, 6, 8, and
24 h), 0.20 mL/time point. Tubes with K2EDTA were used to collect
blood samples. All samples were centrifuged at 6800g at 4 °C for 5
min within 0.5 h and then were stored at −70 °C until LC−MS/MS
analysis. The data on the plasma concentration were analyzed, and the
key pharmacokinetic parameters were obtained.
■
ASSOCIATED CONTENT
* Supporting Information
sı
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.2c00336.
1
H NMR spectra of the products from the reaction of
W1 and 1,4-benzenedithiol; reaction mechanism of W1
with GSH or DMSO-d6 and W8 with GSH; reaction
modeling and resulting energies at the theoretical level;
correlation between GSH t1/2 and LUMO energies; halflife data for reaction of W1 with GSH or DMSO-d6;
9139
https://doi.org/10.1021/acs.jmedchem.2c00336
J. Med. Chem. 2022, 65, 9126−9143
Journal of Medicinal Chemistry
pubs.acs.org/jmc
Lixia Wu − Key Laboratory of Pesticide & Chemical Biology
(CCNU), Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, China
Jiaqi Liu − Key Laboratory of Pesticide & Chemical Biology
(CCNU), Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, China
Chen Su − National Facility for Protein Science in Shanghai,
Zhangjiang Lab, Shanghai 201210, China
Chao Peng − National Facility for Protein Science in
Shanghai, Zhangjiang Lab, Shanghai 201210, China
crystal data and structure refinement for W8; crystallography data collection and refinement statistics of
FBPase−W8 (7WJV); IC50 of W8 against mutants of
FBPase; inhibition of W8 and W8k against BTK/
EGFR/JAK3; LC−MS data of FBPase and W8; ABPPbased proteomic analysis; X-ray cocrystal structures of
leaving groupssaccharin and FBPase; effect of
compounds on blood glucose in the ICR mice model
and glucose output in primary rat hepatocytes; HPLC
analysis data; and 1H NMR, 13C NMR, and 19F NMR
spectra (PDF)
Crystallographic data of W8 (CIF)
Structure of FBPase in a complex with W8 (PDB)
Molecular formula strings (CSV)
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.2c00336
Author Contributions
∥
W.W., H.C., and Y.X. contributed equally to this work. All
authors contributed to the writing of the manuscript and have
approved the final version of the manuscript.
Accession Codes
The atomic coordinates and structure factors have been
deposited into the RCSB Protein Data Bank with accession
number 7WJV. The authors will release the atomic coordinates
and experimental data upon article publication.
■
Article
Funding
This work was supported by the Natural Science Foundation
of China (Nos. 22177036, 21877046, and 21572077), the
Program for PCSIRT (No. IRT0953), the Guizi Scholarship of
CCNU (No. 31101222098), the self-determined research
funds of CCNU from the colleges’ basic research and
operation of MOE (Nos. CCNU19TS011 and
CCNU16A02041), and the support from the Program of
Introducing Talents of Discipline to Universities of China (111
Program, B17019) is also appreciated.
AUTHOR INFORMATION
Corresponding Authors
Yanliang Ren − Key Laboratory of Pesticide & Chemical
Biology (CCNU), Ministry of Education, College of
Chemistry, Central China Normal University, Wuhan
430079, China; orcid.org/0000-0001-8565-2152;
Phone: +86-27-67862022; Email: renyl@ccnu.edu.cn
Yunyuan Huang − Key Laboratory of Pesticide & Chemical
Biology (CCNU), Ministry of Education, College of
Chemistry, Central China Normal University, Wuhan
430079, China; Shanghai Key Laboratory of New Drug
Design, School of Pharmacy, East China University of Science
and Technology, Shanghai 200237, China; Phone: +86-2767862022; Email: huangyy@ecust.edu.cn
Jian Wan − Key Laboratory of Pesticide & Chemical Biology
(CCNU), Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, China;
orcid.org/0000-0003-4172-1392; Phone: +86-2767862022; Email: jianwan@ccnu.edu.cn
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
The authors thank the staff members of the Mass Spectrometry
System at the National Facility for Protein Science in Shanghai
(NFPS), Zhangjiang Lab, China for providing technical
support and assistance in data collection and analysis. The
authors thank the staff of the BL18U1 and BL19U1 beamline
of the NCPSS at the Shanghai Synchrotron Radiation Facility
for assistance in data collection.
■
ABBREVIATIONS USED
ABPP, activity-based protein profiling; ADMET, absorption,
distribution, metabolism, excretion, and toxicity; AMP,
adenosine monophosphate; AUC, area under the curve;
BTK, Bruton’s tyrosine kinase; CA, carbonic anhydrase;
DCM, dichloromethane; DFT, density functional theory;
DMSO, dimethyl sulfoxide; EGFR, epidermal growth factor
receptor; EI, electron ionization; Et3N, triethylamine; FDA,
Food and Drug Administration; FBP, fructose-1,6-diphosphate; FBPase, fructose-1,6-bisphosphatase; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; G3P, glyceraldehyde 3phosphate; GSH, glutathione; HPLC, high-performance liquid
chromatography; HRMS, high-resolution mass spectrometry;
IAA, iodoacetamide alkyne; IC50, half-maximal inhibitory
concentration; ICR, Institute of Cancer Research; JAK3,
Janus kinase 3; LC−MS, liquid chromatography−mass
spectrometry; LUMO, lowest unoccupied molecular orbital;
Met, metformin; MS, mass spectrometry; NADH, nicotinamide adenine dinucleotide; NAS, noncalorie artificial sweetener; NMR, nuclear magnetic resonance; OD, outside
diameter; OGTT, oral glucose tolerance test; QM, quantum
mechanical; SN, nucleophilic substitution; SRR, structure−
reactivity relationship; TCI, targeted covalent inhibitor; TIM,
Authors
Wuqiang Wen − Key Laboratory of Pesticide & Chemical
Biology (CCNU), Ministry of Education, College of
Chemistry, Central China Normal University, Wuhan
430079, China
Hongxuan Cao − Key Laboratory of Pesticide & Chemical
Biology (CCNU), Ministry of Education, College of
Chemistry, Central China Normal University, Wuhan
430079, China
Yixiang Xu − Shanghai Key Laboratory of New Drug Design,
School of Pharmacy, East China University of Science and
Technology, Shanghai 200237, China
Li Rao − Key Laboratory of Pesticide & Chemical Biology
(CCNU), Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, China;
orcid.org/0000-0002-0780-6504
Xubo Shao − Key Laboratory of Pesticide & Chemical Biology
(CCNU), Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, China
Han Chen − Key Laboratory of Pesticide & Chemical Biology
(CCNU), Ministry of Education, College of Chemistry,
Central China Normal University, Wuhan 430079, China
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Journal of Medicinal Chemistry
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